1. Field of the Invention
The present invention is concerned with a new synthesis procedure for preparing novel crosslinked polyamines. The crosslinked polyamines may be used to synthesize new zwitterionic compounds suitable for use as ampholytes in isoelectric focusing of amphoteric substances.
In addition, this invention relates generally to the field of chemical compound analysis. More specifically, a method is described for analysis of carrier ampholytes used in isoelectric focusing. The method includes focusing carrier ampholytes on immobilized ph gradients and then visualizing the ampholytes by precipitation using precipitating solutions such as picric acid.
2. Description of the Related Art
1. Ampholyte Synthesis
Isoelectric focusing ("IEF") is a known process for resolving individual molecular species under either denaturing or non-denaturing conditions. IEF is used to separate, purify, and analyze amphoteric substances such as proteins, enzymes, hormones, antigens, antibodies, etc. IEF is a process wherein an applied electrical field forces heterogeneous carrier zwitterions, zwitterionic mixtures, or ampholytes to segregate into an ordered array of molecules, thus establishing a pH gradient between the anode and cathode electrodes. This procedure can be carried out in either a free solution format or in a gel format.
A zwitterion is a molecule that has at least one region of positive charge and at least one region of negative charge. Ampholytes are substances that may ionize to form either anions or cations. Ampholytes may comprise zwitterions and vice versa. Preferentially, ampholytes used in this invention comprise a mixture of numerous species of zwitterionic chemicals which differ from each other by the nature and number of basic and acidic groups.
Each ampholyte species in an ampholyte mixture has its own intrinsic isoelectric point. In a particularly suitable system of carrier ampholytes, the isoelectric points of the different ampholyte species will cover the pH range 3-10. Most naturally occurring proteins, enzymes, hormones, antigens, antibodies, etc. are isoelectric at some point within this pH range. Substantially uniform distribution of the inherent isoelectric points of the various ampholyte species throughout the desired pH range is an important factor for the formation of uniform and linear pH gradients.
U.S. Pat. Nos. 4,131,534 to Just, 3,901,780 to Denckla, 3,692,654 to Svendsen, and 3,485,736 to Vesterberg all outline various methods for preparing carrier ampholytes.
One well-known method of preparing carrier ampholytes involves synthesizing polyamine-polycarboxylic acid mixtures for use as carrier ampholytes. This method typically utilizes a polyethylene polyamine to which an .alpha.,.beta.-unsaturated acid such as acrylic acid is chemically linked by an addition reaction. The simplest ampholyte formed by that reaction is a polyethylene polyamine molecule to which one carboxylic acid molecule is linked, forming a .beta.-amino carboxylic acid system. Because multiple amino groups are present in the molecule, the addition reaction can occur more than once. Subsequent addition reactions yield .beta.-amino polycarboxylic acids which contain an increasing number of carboxylic acids in the molecule.
As a result of the above synthesis, some of the resulting ampholyte species are obtained in a high yield whereas other species will be formed at the same time in relatively low yield. A suitable ampholyte mixture should preferably exhibit a uniform distribution of buffering capacity and conductivity throughout the pH gradient and preferably provide a large number of buffering species per pH unit. The ampholyte mixture should preferably be evenly distributed across the pH span. The preferable conditions may be achieved when the different ampholyte species are contained in the mixture in approximately equal concentrations. Therefore, at the end of the chemical reaction, fractionation of the ampholyte mixture in expensive multicompartment electrolysis equipment is normally necessary to blend acceptable mixtures by boosting the concentrations of species obtained at low yield in the initial synthesis. A useful ampholyte mixture is then obtained by mixing appropriate amounts of materials from different electrolysis compartments.
The above method of synthesis makes use of known organic chemistry procedures. There are numerous examples in the literature in which nucleophilic substances (such as the amino groups here) react with electron-deficient alkenes (i.e. acrylic acid) to afford addition products. In these instances, it has been demonstrated that the addition reaction yields favorable results when equimolar quantities of each reactant are utilized.
The above method of synthesis involves adding a calibrated amount of aqueous unsaturated carboxylic acid to an aqueous solution of polyethylene polyamine, with simultaneous heating and stirring. Instead of a single amine, amine mixtures may be used. Similarly, instead of a single carboxylic acid, a mixture of several carboxylic acids each containing at least one carbon-carbon double bond within their molecules may be utilized.
Since the reaction between the unsaturated carboxylic acid and the polyethylene polyamine exhibits slow reaction kinetics at room temperature, the reaction is generally performed at elevated temperature. This procedure, however, leads to undesired side products which cause coloration of the product. Additionally, even at elevated temperature, the reaction kinetics are rather slow and it takes several hours for all of the unsaturated carboxylic acid to react.
A problem with the prior art synthesis of carrier ampholyte mixtures is that the starting polyethylene polyamine compounds which are commercially available come in only a limited number of straight chain forms. Thus, the number of different molecular species or isomers of ampholytes that can be made from these is quite limited. Because of all of the above problems, known procedures are not completely satisfactory, and persons skilled in the art have searched for improvements.
2. Ampholyte Analysis
The resolving power of IEF is largely dependent upon the nature of the pH gradient used. Ideally, the pH gradient should be stepless with an even distribution of charged groups throughout the pH range of interest. Immobilized pH gradients ("IPGs") have overcome many of the problems associated with the uneven distribution of charged groups. However, IPGs are generally limited to analytical and small scale preparative procedures using polyacrylamide matrices. In certain applications IPG analysis requires carrier ampholytes to avoid protein-matrix interactions.
In contrast to IPGs, carrier ampholytes ("CA") have the advantage of being used with virtually any IEF support medium (polyacrylamide, agarose, Sephadex, etc.) or with liquid phase preparative equipment. The resolution of a CA gradient is dependent upon the number and quality of the ampholyte species which are used to generate it. The distribution of ampholyte species over a given pH range becomes especially important when considering narrow range preparative or analytical procedures. For research efforts which are aimed at producing more diverse ampholytes it is preferable to have a reproducible method for monitoring the approximate number and linear charge distribution of the ampholyte species which have been synthesized.
Attempts to estimate the total number of molecular species in ampholyte preparations have met with criticism. Some practitioners have emphasized that the absolute number of molecular species in an ampholyte preparation is of limited importance as compared to the buffering capacity of the species which are present. Nevertheless, such methods have been helpful for evaluating the relative heterogeneity of ampholyte species.
One method of estimating the total number of molecular species involves focusing ampholytes in Sephadex (Pharmacia-LKB Biotechnology [LKB], Bromma, Sweden), then rolling a sheet of filter paper saturated with 5% glucose onto the gel surface. The filter paper is then removed and heated at 110.degree. C. to produce visible ampholyte-glucose caramel reaction products. Using this technique, it was estimated that LKB wide range Ampholines contain approximately 62 carrier species. In another method, a paper print of focused ampholytes is treated with formaldehyde, lactose, or ninhydrin. Using this method, wide range LKB Ampholines have been estimated to comprise more than 500 individual species. Discontinuities in ampholyte distribution have been detected by Schlieren patterns and side illumination of focused gels.
Carrier ampholyte heterogeneity has also been studied using methods which attempted to estimate the distribution of conductivity, and the ability of ampholyte preparations to resolve focused proteins. Although the last approach may be the most direct for choosing an ampholyte mixture for separation of specific groups of proteins, its broad application is limited by the lack of a suitable protein preparation which contains a continuum of closely spaced, well-defined protein species.
All of the above-mentioned analytical methods rely on conventional IEF in gradients which are generated by the ampholytes themselves. In contrast, IPG analysis separates ampholytes in a gradient which is dictated by a smooth and continuous distribution of charged groups covalently bound on a gel matrix. Individual ampholyte species migrate to a position in the pH continuum of the IPG which is indicative of their pI.
Prior to this invention, no method known to the inventors has been developed to analyze ampholytes using a constant calibrated medium to compare the heterogeneity of different ampholyte compounds.